Hiv-1 virus tat-protein mutants

ABSTRACT

The invention relates to the use of a protein mutation method for preparing detoxified and immunogenic mutants of the wild-type HIV-1 virus Tat-protein. The invention also relates to a method for preparing detoxified and immunogenic mutants of the wild-type HIV-1 virus Tat-protein, comprising a first step in which wild-type Tat-protein mutants are prepared, a second step in which detoxified mutants having no transcellular activity but an altered nuclear localization are screened, and a third step in which immunogenic mutants capable of inducing antibodies directed against both mutants and the wild Tat protein are screened. Steps two and three are reversible.

A subject of the present invention is HIV-1 virus Tat-protein mutantsand a pharmaceutical composition, in particular a vaccine, comprising atleast one of said mutants.

The HIV virus is the etiological agent of AIDS. The HIV belongs to thefamily of human retroviruses (Retroviridae) and to the sub-family of thelentiviruses. Among the two types of HIV (HIV-1 and HIV-2), HIV-1 is themore cytopathic and the more prevalent world-wide, in particular inWestern countries. HIV-1 infection is accompanied by early dysfunctionof the immune system in humans infected by the virus.

Like the other retroviruses, HIV-1 has genes which code for structuralproteins of the virus. The gag gene codes for the protein which formsthe virion core, including the p24 antigen. The pol gene codes for theenzymes responsible for reverse transcription (reverse transcriptase)and integration (integrase). The env gene codes for the envelopeglycoproteins. However HIV-1 is more complex than the other retrovirusesand contains six other genes (tat, rev, nef, vif, vpr and vpu) whichcode for proteins involved in regulation of the expression of the genesof the virus. The genome of HIV-1 also comprises the 5′ and 3′ LTRs(Long Terminal Repeats) which include regulation elements involved inthe expression of the genes of the virus.

In vivo, Tat is a protein necessary for the replication of HIV-1. Thefunction(s) of Tat in transcription have been much studied and it is nowpretty clear that one of Tat's main roles is the regulation of thetranscription from the 5′ LTR. Tat is an activator of transcription bytransactivation of the 5′ LTR, via its fixation to the TAR sequence atthe same time as to other cell factors, resulting in an increase inviral transcription and elongation. The transactivation of the LTR bythe Tat protein is essential both for the expression of the genes andthe replication of the virus. The transactivation of the viral promoterby the Tat protein (17, 18) allows the large-scale production of viralmessenger RNAs the transfer of which into the cytoplasm depends onanother regulatory protein, the Rev protein. Tat and Rev regulate theexpression of HIV-1 (7). The Tat protein is secreted by cells infectedby HIV-1. Once outside the cell, it is capable of being internalized byneighbouring cells (9, 14), infected or not, thus being able to inducemodifications to the state of activation of non-infected T lymphocytes.It is therefore directly involved in the progression of AIDS andprobably in pathologies associated with AIDS, such as Kaposi's sarcoma.

The complete Tat protein is made up of 101 amino acids, residues 1-72being coded by a first exon and residues 73-101 being coded by a secondexon. The Tat protein is strongly conserved. A truncated form of 86amino acids, which does not correspond to the native form, exists in afew laboratory strains obtained after culture passages. This truncatedform is due to the introduction of a stop codon at position 87 duringthe culture passages, but more than 90% of the Tat proteins studiedmaintain the configuration of 101 amino acids. Although amino acids87-101 could not contribute greatly to ex vivo propagation, theirconservation in the natural isolates of HIV-1 which replicate is anindication of their biological importance. HIV-1's native Tat protein of101 amino acids is made up of five physical domains, but the molecularmechanism by which it acts has not yet been completely explained.Briefly, these five domains are described in the publication of Jeang,K. T et al. (18). In this publication, domain 1 corresponds to aminoacids 1-20 which are rich in acid residues, domain 2 corresponds toamino acids 21-40 which are rich in cysteine residues (7 cysteineresidues, 6 of which are very strongly conserved), domain 3 correspondsto amino acids 41-48 and contains the RKGLGI motif common to HIV-1,HIV-2 and SIV, domain 4 corresponds to amino acids 49-72 and contains abasic RKKRRQRRR motif and domain 5 corresponds to amino acids 73-101 andcomprises an RGD motif. The role of domain 1 has not yet been explained.It has only been shown that changes in a single amino acid in thisdomain were well tolerated and did not alter the functionality of theTat protein. One hypothesis put forward is that domain 1 could beinvolved in transactivation. Changing six cysteines out of the seven indomain 2 suppresses the functionality of the Tat protein. This domain isimportant for transactivation. The role of domain 3 has not beenelucidated. Domain 4 confers the properties of Tat fixation on the TARRNA and is important for nuclear localization as well as fortranscellular transport of the Tat protein. Domain 5 would also beinvolved in the transcellular transport of the Tat protein.

In the detailed description of the invention which follows, the presentinventors started from the sequence of the ACH320.2A.2.1 strain (NCBIaccession no. U34604) and refined the notion of domains given in thepublication of Jeang, K. T et al. (18). Thus and with reference to thisparticular strain, in the present invention domain 1 corresponds toamino acids 1-21 (role not elucidated), domain 2 corresponds to aminoacids 22-37 (involved in transactivation), domain 3 corresponds to aminoacids 38-48 (role unknown) and domain 5 corresponds to amino acids73-101 (transcellular transport). In domain 4 corresponding to aminoacids 49-72, it is the peptide 49-57 which is important for fixation tothe TAR RNA, for nuclear localization and for the transcellulartransport of Tat (18).

The development of a vaccine against HIV-1 is awaited world-wide. Inpatients infected with HIV-1, an immune response to Tat and Rev isdetected only in individuals for whom the infection does not progress toAIDS. (26). Several studies of vaccination using Tat and/or Rev in theSIV animal model have shown a partial or complete protection againstinfection (4-6, 21). However, direct transposition of these vaccinationprotocols to humans is not possible. It has been shown inter alia thatTat has toxic effects in vitro (19, 22). These toxic effects include (i)deregulation of cell signals involved in apoptosis (28, 30), (ii)deregulation of the expression of parts of genes of the immune systemsuch as the gene coding for interleukin-2 (29), or genes coding for themolecules of the major histocompatibility complex (MHC) of class I (16),and/or (iii) induction of angiogenesis (1, 2, 20). The Tat protein musttherefore be detoxified before being used as a vaccine antigen. One teamchose to detoxify the Tat protein by chemical inactivation (10).However, such inactivation can be carried out only with a view to usingrecombinant proteins as vaccine antigens. In order to be able to use theTat protein in the form of nucleic acid in a recombinant vector, livingor not, only a genetic detoxification can be envisaged. The presentinventors therefore chose to explore this route for the detoxificationof the Tat protein, by directed mutagenesis in order to allow its useboth as a vaccine protein sub-unit and/or as part of a vaccinationvector.

The present invention relates to the use of a protein mutation processfor the preparation of detoxified and immunogenic wild-type Tat-proteinmutants.

By wild-type Tat protein “mutants” is meant mutants obtained bysubstitution or replacement of one or more amino acids.

By “detoxified wild-type Tat-protein mutants” is meant a Tat protein nolonger having the following toxic effects:

-   -   when it is secreted by an infected cell, the Tat protein is        toxic in exogenous form to cells not infected with HIV-1, due to        its capacity to induce a cell signalization by fixation to        surface receptors, and its ability to be internalized by        non-infected cells and transported to the nucleus of the target        cell;    -   in exogenous and endogenous manner, the Tat protein will be        localized in the nucleus of the target cell and induce the        regulation of the expression of cell genes, being able to        involve the transactivating properties or domain 5 of the Tat        protein.

By “immunogenic wild-type Tat-protein mutants” is meant a mutant capableof inducing the production of antibodies after injection into a modelanimal, these antibodies having the capacity to react both with theTat-protein mutant but also with the wild-type Tat protein.

The invention also relates to a process for preparing detoxified andimmunogenic wild-type Tat-protein mutants characterized in that itcomprises:

-   -   a stage of preparation of wild-type Tat-protein mutants, in        particular by mutation of the nucleic acid coding for the        wild-type Tat protein,    -   a stage of screening the detoxified mutants characterized by an        absence of transcellular activity and an alteration of the        nuclear localization, and optionally by an absence of        transactivating activity, and    -   a stage of screening the immunogenic mutants characterized by        their ability to induce antibodies directed against both said        mutants and the wild-type Tat protein, the order of the last two        stages being reversible.

The absence of transcellular activity also means an absence oftranscellular transport and can be detected in a cell line establishedby the absence of activation of the viral promotor of the LTR-reportergene construction, for example that of chloramphenicol acetyltransferase(CAT), the expression of which depends on the viral promoter (LTR) in anestablished cell line (31), after these cells are brought into contactwith a Tat-protein mutant produced in exogenous manner, for example by acell line other than that containing the reporter gene dependent on theviral LTR (32).

The alteration of the nuclear localization can be defined as thepresence of the Tat protein in the cytoplasmic compartment of cellstransfected by nucleic acids coding for the wild-type Tat-proteinmutants, for example in the 72 hours following transfection, and can bedetected either by optical microscopy after transfection of cell linesby the nucleic acids coding for the Tat-protein mutants by techniquesinvolving immunomarking of the product of these genes, or by thedetection after transfection of the product of the translation ofnucleic acids containing the gene coding for a Tat mutant fused to thegene coding for an autofluorescent protein such as the EGFP protein (33,34).

The absence of transactivating activity corresponds to the absence ofactivation of the viral promoter and can be detected by the absence ofexpression of a reporter gene, for example that of chloramphenicolacetyltransferase (CAT), the expression of which depends on the viralpromoter (LTR) in an established cell line, after transfection of thisline by nucleic acids coding for the wild-type Tat-protein mutants (31).

The invention also relates to a detoxified and immunogenic mutant of theTat protein of the HIV-1 virus, characterized in that it comprises atleast two mutations in regions 4 and/or 5 of the wild-type Tat protein,and in that, when the mutation is in the region of domain 4, it is inthe part delimited by the amino acid in position 49 to the amino acid inposition 57, and in that, when the mutation is in domain 5, it is eitherin the RGD motif, or in the region 88-92, preferably in positions 89and/or 92, the mutations being mutations by substitution of one aminoacid by another.

An advantageous mutant according to the present invention is a mutant asdefined above, characterized in that it comprises at least one mutationin region 4.

The invention also relates to a mutant as defined above, characterizedin that the mutations in domains 4 and/or 5 are capable of conferring atleast one of the following properties:

-   -   the cancellation of the transcellular effect of the wild-type        Tat protein,    -   the alteration of the nuclear localization of the wild-type Tat        protein.

The invention relates to a mutant as defined above, characterized inthat it comprises an additional mutation capable of conferring a loss ofthe transactivating activity of the wild-type Tat protein.

A Tat protein usable as vaccination antigen will therefore have to meetmost of the following criteria:

-   -   cancellation of the transcellular effect of Tat (domain 4 and/or        5)    -   alteration of the nuclear localization of Tat (domain 4)    -   loss of the transactivating activity (domain 2)    -   maintenance of the protein's antigenicity (a maximum of 4 or 5        mutations, modifying the CTL epitopes as little as possible)

According to an advantageous embodiment, the present invention relatesto a mutant as defined above, characterized in that it comprises amutation in the N-terminal region of domain 4 of the wild-type Tatprotein, in particular in the part delimited by the amino acid inposition 49 to the amino acid in position 57.

An advantageous mutant according to the invention is a mutant as definedabove, characterized in that it comprises a mutation in the N-terminalregion of domain 4 of the wild-type Tat protein in the part delimited bythe amino acid in position 49 to the amino acid in position 55.

An advantageous mutant according to the invention is a mutant as definedabove, characterized in that it comprises a mutation in at least one ofthe following regions in domain 5 of the wild-type Tat protein:

-   -   the RGD motif,    -   the region 88-92, preferably in positions 89 and/or 92.

An advantageous mutant according to the invention is a mutant as definedabove, characterized in that it comprises a mutation in domain 2 of thewild-type Tat protein, in particular the replacement of any one of thecysteines, advantageously by a serine.

The invention also relates to a mutant as defined above, characterizedin that it comprises at least one of the following mutations:

-   -   replacement in position 27 of a cysteine by a serine,    -   replacement in position 51 of a lysine by a threonine,    -   replacement in position 52 of an arginine by a leucine,    -   replacement in position 55 of an arginine by a leucine,    -   replacement in position 57 of an arginine by a leucine,    -   replacement in position 79 of a glycine by an alanine,    -   replacement in position 89 of a lysine by a leucine,    -   replacement in position 92 of a glutamic acid by a glutamine.

The invention also relates to a mutant as defined above, characterizedin that it is chosen from the mutants having two mutations as indicatedhereafter, each of the mutations being represented by a triplet:letter-figure-letter, the figure of which indicates the position of themutated amino acid, the letter preceding the figure corresponds to theamino acid to which the mutation relates and the letter following thefigure corresponds to the amino acid replacing the amino acid precedingthe figure: K51T-R52L (SEQ ID NO: 2) K51T-R55L (SEQ ID NO: 3) K51T-R57L(SEQ ID NO: 4) K51T-G79A (SEQ ID NO: 5) K51T-K89L (SEQ ID NO: 6)K51T-E92Q (SEQ ID NO: 7) R52L-R55L (SEQ ID NO: 8) R52L-R57L (SEQ ID NO:9) R52L-G79A (SEQ ID NO: 10) R52L-K89L (SEQ ID NO: 11) R52L-E92Q (SEQ IDNO: 12) R55L-R57L (SEQ ID NO: 13) R55L-G79A (SEQ ID NO: 14) R55L-K89L(SEQ ID NO: 15) R55L-E92Q (SEQ ID NO: 16) R57L-G79A (SEQ ID NO: 17)R57L-K89L (SEQ ID NO: 18) R57L-E92Q (SEQ ID NO: 19) G79A-K89L (SEQ IDNO: 20) G79A-E92Q (SEQ ID NO: 21) K89L-E92Q (SEQ ID NO: 22)

An advantageous mutant according to the present invention is a mutant asdefined above, characterized in that it is chosen from the followingmutants: K51T-R55L (SEQ ID NO: 3) R52L-R55L (SEQ ID NO: 8) R52L-G79A(SEQ ID NO: 10) R55L-R57L (SEQ ID NO: 13) G79A-K89L (SEQ ID NO: 20)

The invention also relates to a mutant as defined above, characterizedin that it is chosen from the mutants having three mutations asindicated hereafter, each of the mutations being represented by atriplet: letter-figure-letter, the figure of which indicates theposition of the mutated amino acid, the letter preceding the figurecorresponds to the amino acid to which the mutation relates and theletter following the figure corresponds to the amino acid replacing theamino acid preceding the figure: C27S-K51T-R52L (SEQ ID NO: 23)C27S-K51T-R55L (SEQ ID NO: 24) C27S-K51T-R57L (SEQ ID NO: 25)C27S-K51T-G79A (SEQ ID NO: 26) C27S-K51T-K89L (SEQ ID NO: 27)C27S-K51T-E92Q (SEQ ID NO: 28) C27S-R52L-R55L (SEQ ID NO: 29)C27S-R52L-R57L (SEQ ID NO: 30) C27S-R52L-G79A (SEQ ID NO: 31)C27S-R52L-K89L (SEQ ID NO: 32) C27S-R52L-E92Q (SEQ ID NO: 33)C27S-R55L-R57L (SEQ ID NO: 34) C27S-R55L-G79A (SEQ ID NO: 35)C27S-R55L-K89L (SEQ ID NO: 36) C27S-R55L-E92Q (SEQ ID NO: 37)C27S-R57L-G79A (SEQ ID NO: 38) C27S-R57L-K89L (SEQ ID NO: 39)C27S-R57L-E92Q (SEQ ID NO: 40) C27S-G79A-K89L (SEQ ID NO: 41)C27S-G79A-E92Q (SEQ ID NO: 42) C27S-K89L-E92Q (SEQ ID NO: 43)

The present invention also relates to a mutant as defined above,characterized in that it is chosen from the following mutants:C27S-K51T-R55L (SEQ ID NO: 24) C27S-R52L-R55L (SEQ ID NO: 29)C27S-R52L-G79A (SEQ ID NO: 31)

The present invention relates to a mutant as defined above,characterized in that it is chosen from the mutants having fourmutations as indicated hereafter, each of the mutations beingrepresented by a triplet: letter-figure-letter, the figure of whichindicates the position of the mutated amino acid, the letter precedingthe figure corresponds to the amino acid to which the mutation relatesand the letter following the figure corresponds to the amino acidreplacing the amino acid preceding the figure: C27S-K51T-R52L-G79A (SEQID NO: 44) C27S-K51T-R52L-K89L (SEQ ID NO: 45) C27S-K51T-R52L-E92Q (SEQID NO: 46) C27S-K51T-R55L-G79A (SEQ ID NO: 47) C27S-K51T-R55L-K89L (SEQID NO: 48) C27S-K51T-R55L-E92Q (SEQ ID NO: 49) C27S-K51T-R57L-G79A (SEQID NO: 50) C27S-K51T-R57L-K89L (SEQ ID NO: 51) C27S-K51T-R57L-E92Q (SEQID NO: 52) C27S-K51T-G79A-K89L (SEQ ID NO: 53) C27S-K51T-G79A-E92Q (SEQID NO: 54) C27S-K51T-K89L-E92Q (SEQ ID NO: 55) C27S-R52L-G79A-K89L (SEQID NO: 56) C27S-R52L-G79A-E92Q (SEQ ID NO: 57) C27S-R52L-K89L-E92Q (SEQID NO: 58) C27S-R52L-R55L-G79A (SEQ ID NO: 59) C27S-R52L-R55L-K89L (SEQID NO: 60) C27S-R52L-R55L-E92Q (SEQ ID NO: 61) C27S-R52L-R57L-G79A (SEQID NO: 62) C27S-R52L-R57L-K89L (SEQ ID NO: 63) C27S-R52L-R57L-E92Q (SEQID NO: 64) C27S-R55L-G79A-K89L (SEQ ID NO: 65) C27S-R55L-G79A-E92Q (SEQID NO: 66) C27S-R55L-K89L-E92Q (SEQ ID NO: 67) C27S-R55L-R57L-G79A (SEQID NO: 68) C27S-R55L-R57L-K89L (SEQ ID NO: 69) C27S-R55L-R57L-E92Q (SEQID NO: 70) C27S-R57L-G79A-K89L (SEQ ID NO: 71) C27S-R57L-G79A-E92Q (SEQID NO: 72) C27S-R57L-K89L-E92Q (SEQ ID NO: 73) C27S-G79A-K89L-E92Q (SEQID NO: 74)

An advantageous mutant according to the present invention ischaracterized in that it is chosen from the following mutants:C27S-K51T-R55L-G79A (SEQ ID NO: 47) C27S-K51T-R55L-K89L (SEQ ID NO: 48)C27S-K51T-R55L-E92Q (SEQ ID NO: 49) C27S-R52L-R55L-G79A (SEQ ID NO: 59)

The present invention relates to a mutant as defined above,characterized in that it is chosen from the mutants having fivemutations as indicated hereafter, each of the mutations beingrepresented by a triplet: letter-figure-letter, the figure of whichindicates the position of the mutated amino acid, the letter precedingthe figure corresponds to the amino acid to which the mutation relatesand the letter following the figure corresponds to the amino acidreplacing the amino acid preceding the figure: C27S-K51T-G79A-K89L-E92Q(SEQ ID NO: 75) C27S-K51T-R52L-R55L-G79A (SEQ ID NO: 76)C27S-K51T-R52L-R55L-K89L (SEQ ID NO: 77) C27S-K51T-R52L-R55L-E92Q (SEQID NO: 78) C27S-K51T-R52L-R57L-G79A (SEQ ID NO: 79)C27S-K51T-R52L-R57L-K89L (SEQ ID NO: 80) C27S-K51T-R52L-R57L-E92Q (SEQID NO: 81) C27S-K51T-R52L-G79A-K89L (SEQ ID NO: 82)C27S-K51T-R52L-G79A-E92Q (SEQ ID NO: 83) C27S-K51T-R52L-K89L-E92Q (SEQID NO: 84) C27S-K51T-R55L-R57L-G79A (SEQ ID NO: 85)C27S-K51T-R55L-R57L-K89L (SEQ ID NO: 86) C27S-K51T-R55L-R57L-E92Q (SEQID NO: 87) C27S-K51T-R55L-G79A-K89L (SEQ ID NO: 88)C27S-K51T-R55L-G79A-E92Q (SEQ ID NO: 89) C27S-K51T-R55L-K89L-E92Q (SEQID NO: 90) C27S-K51T-R57L-G79A-K89L (SEQ ID NO: 91)C27S-K51T-R57L-G79A-E92Q (SEQ ID NO: 92) C27S-K51T-R57L-K89L-E92Q (SEQID NO: 93) C27S-R52L-R55L-R57L-G79A (SEQ ID NO: 94)C27S-R52L-R55L-R57L-K89L (SEQ ID NO: 95) C27S-R52L-R55L-R57L-E92Q (SEQID NO: 96) C27S-R52L-R55L-G79A-K89L (SEQ ID NO: 97)C27S-R52L-R55L-G79A-E92Q (SEQ ID NO: 98) C27S-R52L-R55L-K89L-E92Q (SEQID NO: 99) C27S-R52L-R57L-G79A-K89L (SEQ ID NO: 100)C27S-R52L-R57L-G79A-E92Q (SEQ ID NO: 101) C27S-R52L-R57L-K89L-E92Q (SEQID NO: 102) C27S-R52L-G79A-K89L-E92Q (SEQ ID NO: 103)C27S-R55L-R57L-G79A-K89L (SEQ ID NO: 104) C27S-R55L-R57L-G79A-E92Q (SEQID NO: 105) C27S-R55L-R57L-K89L-E92Q (SEQ ID NO: 106)C27S-R55L-G79A-K89L-E92Q (SEQ ID NO: 107) C27S-R57L-G79A-K89L-E92Q (SEQID NO: 108)

An advantageous mutant according to the invention is a mutant as definedabove, characterized in that it is chosen from the following mutants:C27S-K51T-R55L-G79A-K89L (SEQ ID NO: 88) C27S-K51T-R55L-G79A-E92Q (SEQID NO: 89)

The present invention also relates to nucleotide sequences coding forone of the mutants as defined above.

The present invention also relates to the cell lines transfected with anucleotide sequence of the invention.

The invention also relates to antibodies directed against one of themutants as defined above, and not recognizing domain D1 of the wild-typeprotein.

Such antibodies are selected by testing and eliminating those whichpossess an affinity for a peptide corresponding to domain D1 of Tat,containing at least the sequence EPVDPKLEPWKHPGS (residues 2-16), forexample in an Elisa format test.

The antibodies according to the invention do or do not recognize thewild-type protein.

An advantageous class of antibodies according to the invention comprisesthe antibodies as defined above, recognizing the wild-type protein.

The antibodies according to the invention are polyclonal or monoclonalantibodies.

The abovementioned polyclonal antibodies are obtained by immunization ofan animal with at least one mutant according to the invention, followedby recovery of the sought antibodies in purified form, by taking asample of the serum of said animal, and separation of said antibodiesfrom the other constituents of the serum, in particular by affinitychromatography over a column on which is fixed an antigen specificallyrecognized by the antibodies, in particular a mutant according to theinvention.

The monoclonal antibodies according to the invention can be obtained bythe hybridomas technique the general principle of which is recalledhereafter.

In a first phase, an animal, generally a mouse (or cells in culturewithin the framework of immunizations in vitro), is immunized with amutant according to the invention, the B lymphocytes of which are thencapable of producing antibodies to said mutant. These antibody-producinglymphocytes are then fused with “immortal” myelomatous cells (murine inthe example) in order to produce hybridomas. From the thus-obtainedheterogeneous mixture of cells, a selection is then made of cellscapable of producing a particular antibody and multiplying indefinitely.Each hybridoma is multiplied in clone form, each leading to theproduction of a monoclonal antibody the recognition properties of whichvis-a-vis the mutant of the invention can be tested for example byELISA, by immunotransfer in one or two dimensions, byimmunofluorescence, or using a biocaptor. The monoclonal antibodies thusselected are subsequently purified in particular according to theaffinity chromatography technique described above.

The present invention also relates to a pharmaceutical composition, inparticular a vaccine, containing as active ingredient at least one ofthe mutants as defined above or at least one of the nucleotide sequencesas defined above, placed under the control of elements necessary to aconstitutive expression of one of the mutants as defined above or atleast one of the antibodies as defined above, in combination with apharmaceutically suitable vehicle.

Of course, a person skilled in the art will easily determine thequantity of mutant to be used as a function of the constituents of thepharmaceutical composition.

The present invention also relates to a diagnostic composition for thedetection and/or quantification of the HIV-1 virus comprising at leastone mutant as defined above, or at least one antibody as defined above.

Of course, a person skilled in the art will easily determine thequantity of mutant to be used as a function of the diagnostic techniqueused.

The invention also relates to a process for the detection and/orquantification of the HIV-1 virus in a biological sample taken from anindividual capable of being infected with HIV-1, such as plasma, serumor tissue, characterized in that it comprises stages consisting of:

-   -   bringing said biological sample into contact with a diagnostic        composition comprising a mutant as defined above or an antibody        as defined above, under predetermined conditions which allow, if        necessary, the formation of antibody/antigen complexes between        the mutant defined above and antibodies directed against the        wild-type Tat protein or between the antibodies defined above        and the wild-type Tat protein, and    -   detecting and/or quantifying the formation of said complexes by        any appropriate means.

The processes of detection and/or of quantification of the virus areimplemented using standard techniques well known to a person skilled inthe art and there can be mentioned, by way of illustration, blots,so-called sandwich techniques and competition techniques.

The invention also relates to the use of at least one mutant as definedabove or at least one antibody as defined above for the in vitrodiagnosis of the HIV-1 virus in a biological specimen or sample.

The invention also relates to the use of at least one mutant as definedabove or at least one antibody as defined above for the preparation of avaccine composition.

The inventors thus showed that for the abovementioned uses it wasnecessary to carry out at least one mutation at domain 4 and/or at leastone mutation at domain 5 of the Tat protein. They obtained, by directedmutagenesis, Tat-protein mutants which were then selected according totheir properties. The mutants retained are chosen from the mutantshaving at least one of the following mutations: K51T (replacement inposition 51 of a lysine by a threonine at domain 4), R52L (replacementof an arginine by a leucine in position 52 in domain 4), R55L(replacement of an arginine by a leucine in position 55 in domain 4),R57L (replacement of an arginine by a leucine in position 57 in domain4), G79A (replacement of a glycine by an alanine in position 79 indomain 5), K89L (replacement of a lysine by a leucine in position 89 indomain 5) and E92Q (replacement of a glutamic acid by a glutamine inposition 92 in domain 5). All the amino acid positions described aboveand subsequently are given with reference to the complete sequence of101 amino acids of the strain ACH320.2A.2.1. A subject of the inventionis the abovementioned mutants. But the invention also relates to mutantshaving two mutations at domain 4 of the Tat protein. These “double”mutants are selected from the mutants K51T-R55L, R52L-R55L, R52L-G79A,R55L-R57L and G79A-K89L. The inventors then showed that by combiningthese double mutations in domain 4 with an additional C27S mutation indomain 2 (replacement of a cysteine by a serine), they obtained verysatisfactory results. Thus, the invention also covers a mutant chosenfrom the “triple” mutants C27S-K51T-R55L, C27S-R52L-R55L andC27S-R52L-G79A. Preferably, the mutant chosen is the mutantC27S-K51T-R55L. Finally, they proved that a “quadruple” mutant combiningat least one mutation in domain 2, two mutations in domain 4 and atleast one mutation in domain 5 had excellent performances for obtaininga non-toxic Tat protein. The “quadruple” mutant is chosen from themutants C27S-K51T-R55L-G79A, C27S-K51T-R55L-K89L, C27S-K51T-R55L-E92Qand C27S-R52L-R55L-G79A. Preferably, the mutant chosen is the mutantC27S-K51T-R55L-G79A.

DESCRIPTION OF FIGURES

FIG. 1 represents a diagrammatic representation of the PCR-directedpoint mutagenesis technique.

FIG. 2 represents the alignment of the protein sequence of the Tatprotein of the strain ACH320.2A.2.1 (SEQ ID NO: 1) and of the proteinsequences of the mutated Tat proteins of the invention.

FIGS. 3 a and 3 b correspond to a diagram representing thetransactivating capacity of ACH320.2A.2.1 or its mutants. The resultsrepresented for each construction correspond to the average of twoindependent experiments. In FIG. 3 a, the NT (non-transfected) columnrepresents the basal activity of the LTR-CAT construction.

The y-axis represents the transactivation multiplication factor.

FIGS. 4 a and 4 b represent the intracellular localization of theACH320.2A.2.1 construction or its mutants.

FIG. 5 represents the transduction capacity of the ACH320.2A.2.1construction or its mutants. The values indicated correspond to theaverage of two independent experiments. The cleavage line was determinedby calculating the average +3 SD (standard deviation) of thetransactivation percentage measured for pEGFP.

The white columns correspond to a 293T/HL3T1 co-culture and the blackcolumns to the transfection of the HL3T1 cells.

The y-axis corresponds to the transactivation percentage relative towild-type ACH320.2A.2.1.

FIG. 6 represents an additional method for screening the cell linesexpressing the mutants of the invention (described in Example 5).

Table 1 represents the oligonucleotides used for the PCR-directedmutagenesis of Tat.

EXAMPLE 1 Construction of the Mutated DNA Coding for the Mutated TatProtein

A cDNA fragment comprising 306 base pairs corresponding to the two exonsof the wild-type Tat gene of the isolate ACH320.2A.2.1 of HIV-1 (11, 12)is mutated using a commercial PCR kit (Clontech) and the nucleotideprimers described in Table 1. The principle of the PCR-directed pointmutagenesis is described in FIG. 1.

As shown in FIG. 1, starting from the cDNA of the wild-type tat gene,two PCRs are carried out independently with a primer situated at the end(E5′ or E3′) and an internal primer situated in the gene and carryingthe desired mutation (M3′ and M5′, respectively) (first PCR cycle). Thetwo PCR products are then mixed in equimolar manner and a second PCRcycle is carried out with end primers containing the restriction sitesfor EcoRI at 5′ and SaII at 3′. Thus the cDNAs are obtained mutated atthe desired point.

This principle was used for all the mutants except for the K89L andE92Q. mutations For the latter the nucleotides having to be mutated werelocalized in the proximity of one of the ends of the cDNA, allowing adirect semi-nested PCR-directed mutagenesis using for the first PCRcycle respectively the following pairs of primers: E5′/K89L (M3′) andE5′/E92Q (M5′).

The double mutant R52L-R55L was generated using the following primersfor single mutagenesis containing the two mutations: R52L-R55L (M5′)5′-GGCAGGAAGCTTAGACAGCTGCGAAGATC-3′ R52L-R55L (M3′)5′-GATCTTCGCAGCTGTCTAAGCTTCTTCCTGCC-3′

The double mutant R52L-G79A was obtained using as matrix the cDNA of themutant G79A and as a primer pair the pair R52L (M5′) /R52L (M3′) for thePCR-directed mutagenesis.

The double mutant G79A-K89L was generated by semi-nested PCR using asmatrix the cDNA of the mutant G79A and the primer pair E5′/K89L (M3′)for the first PCR cycle.

The triple mutant C27S-K51T-R55L (STL) was obtained using the cDNA ofthe double mutant K51T-R55L as matrix and a primer pair C27S (M5′)/C27S(M3′) for the PCR-directed mutagenesis.

The triple mutant C27S-R52L-G79A was obtained using as matrix the cDNAof the mutant R52L-G79A and as primer pair the pair C27S (M5′)/C27S(M3′) for the PCR-directed mutagenesis.

The quadruple mutant C27S-K51T-R55L-G79A (STLA) was generated using thecDNA of STL as matrix and the primers G79A (M5′)/G79A (M3′) for themutagenesis.

All the first PCR cycles were carried out using 0.5 μg of plasmid, 0.29ng/ml of each of the primers under the following conditions: 1×94° C. 5′1×[94° C. 2′50° C. 2′ 72° C. 4′] 25×[94° C. 1′ 50° C. 1′ 72° C. 4′]1×72° C. 5′.

The primer pair EcoRI/SalI was used for the second cycle of all thePCR-directed mutageneses, except for the mutants R52L, R55L and thedouble mutant R52L-R55L, for which the primer SalI/3′ was replaced bythe primer E6854. In all cases the second cycle was carried out usingthe same conditions as for the first cycle and 0.5 μl of the products ofPCR 5′ and 3′ of the first cycles (FIG. 1). A strip of 323 base pairswas created and was then bound in the pCR2,1-Topo (Invitrogen, K4500-40)plasmid according to the manufacturer's protocol in order to create thepCR-TEX constructions.

The positive clones were then selected after automated sequencing usingthe DyeTerminator (trade name) sequencing mix on the 377X automaticsequencer (Applied Biosystems). Analysis of the sequences was carriedout using the MacVector 7.0 software (Oxford Molecular). Among all theconstructions sequenced, all contain only the desired mutation, exceptone of the clones derived from the product PCR-RS55L, which showed anadditional mutation K→T at position 51. This double mutant K51T-R55L wastherefore conserved for additional analysis, and the single mutant K51Twas generated using the primers K51T (M5′) and K51T (M3′).

The EcoRI-SalI or EcoRI-EcoRI fragments of the pCR-TEX constructionswere sub-cloned in a eukaryotic vector pEGFP-C2 (Clontech) in which theTat mutant is fused in the C-terminal position with EGFP (EnhancedFluorescent Green Protein). It was previously shown that the fusion ofthe EGFP to Tat does not alter Tat's transactivating capacity, nor itscellular localization (25). The cloning stages were carried out inEscherichia coli (E. Coli) DH5α according to standard molecular biologytechniques (23). The pEGFP-TEX constructions, in which the expression ofthe fusion protein is under the control of the Cytomegalovirus (CMV)promoter, were obtained and screened by automatic sequencing forpositive clones. The amino acid sequences of the positive clones whichwere selected are represented in FIG. 2. The DNA of these clones wasamplified, purified using the Nucleobond AX kit (trade name)(Macherey-Nagel), according to the manufacturer's instructions.

EXAMPLE 2 Transactivating Capacity of the Tat Mutants

In order to study the transactivating capacity of the EGFP-Tat fusionproteins, the cell line HL3T1 was used. This line is a HeLa cellderivative, transfected in stable manner with a chloramphenicolacetyltransferase (CAT) gene dependent on the viral HIV-1 promoter (LTR)(8). One day after seeding of 2.5×10⁵ HL3T1 cells in 6-well plates, thecells were transfected with 2 μg of the pEGFP-TEX constructions usingthe Exgen 500 kit (marketed by Euromedex) according to the protocolrecommended by the manufacturer. After 48 hours to 72 hours of culture,the cells were trypsinized and the quantity of transfected cells wasestimated by fluorescence microscopy. The equivalent of 1.5×10³fluorescent cells was lysed in 100 μl of Tris 0.01M-EDTA 1 nM-NaCl 150mM (TEN) and subjected to a freezing/thawing stage before treatment for20 minutes at 65° C. The CAT activity was then measured in aphase-extraction test, as previously described (24). Briefly, 70 μl ofcell lysates were incubated for 2 hours at 37° C. with 130 μl of the CATreaction mixture (Tris-HCl pH=7.5 150 mM, EDTA 0.2 nM, NaCl 30 mM,butyryl coenzyme A 0.3 mg/ml, glycerol 3%,D-threo-[dichloroacetyl-1-¹⁴C]chloramphenicol 0.08 μCi.). The reactionmixture was then extracted using 400 μl of a mixture of pristane(2,6,10,14-tetramethylpentadecane) and xylene in a volume/volume ratioof 2:1. The radioactivity was measured on 300 μl of the resultingorganic phase using a scintillation counter.

FIGS. 3 a and 3 b show that none of the single mutations alone allowsthe complete destruction of the transactivating activity of the Tatprotein of ACH320.2A.2.1, except for the mutant C27S. The mutant R55Ldoes not very significantly modify the transactivating activity ofACH320.2A.2.1 Tat. But this mutation combined with one of the mutationsK51T or R52L shows a very significant inhibition of the transactivatingactivity of Tat. This inhibition is total for the triple and quadruplemutants STL and STLA.

EXAMPLE 3 Intracellular Localization of the Tat-EGFP Fusion Proteins

The basic Tat domain is responsible for the nuclear localization of Tat.Certain of the mutations which were constructed affect this basicdomain. Also, the inventors wanted to identify the mutations whichaffected the intracellular localization of Tat. After seeding of 2.5×10⁵HL3T1 cells on microscope slides, the cells were transfected with 2 μgof each pEGFP-TEX construction using the Exgen 500 kit (marketed byEuromedex) according to the protocol recommended by the manufacturer.After 1, 2 or 3 days the slides are recovered and fixed with 4%paraformaldehyde before observation using an Axioplan 2 (trade name)fluorescence microscope (Zeiss). As shown in FIG. 4 a or b (A and B) andas already described (25), the wild-type Tat-EGFP fusion protein showeda nuclear localization after 3 days of culture. The single Tat mutationsdid not affect this localization (FIG. 4 a, C to H), nor the doublemutation R52L-R55L (FIG. 4 a, I), R52L-G79A (FIG. 4 b, C), G79A-K89L(FIG. 4 b, D), nor the triple mutant C27S-R52L-G79A (FIG. 4 b, E).However, the K51T-R55L combination or multiple mutants containing it(STL, STLA) showed both a nuclear and cytoplasmic localization of theTat protein-EGFP on the 3rd day (FIG. 4 a, J to L), whereas the signalwas strictly nuclear on the 1st and 2nd day following the transfection.It therefore seems that Tat's nuclear localization signal isdiscontinuous and contains at least the residues K51 and R55, but notR52.

EXAMPLE 4 Transcellular Activity of the Tat Mutants

Different studies have shown that the total number of basic residues inthe basic Tat domain plays a role in the capacity of the Tat protein,secreted by infected cells present in the extracellular medium, to beinternalized by non-infected cells, a phenomenon also calledtransduction. The inventors evaluated the transduction capacity of theconstructions containing mutations in this basic domain. A co-culturetest was carried out between producer cells and effector cells. The 293Tcells were transfected with 3 μg of the pEGFP-TEX constructions usingthe calcium phosphate technique (23). 24 hours after transfection, thetransfected 293T cells were trypsinized and co-cultivated with 2.5×10⁵HL3T1 cells for another 48 hours in the presence of 100 μMchloroquinine. The cells were then harvested and lysed in TEN beforeevaluation of the CAT activity, as described previously. Because thissystem depends on both the effectiveness of the transduction and thetransactivating activity, the inventors started from the premise that adrastic change in the transduction capacity would be reflected in asignificant difference between the activity measured after directtransfection and the activity measured after co-culture, compared with astandardized positive control. This is the reason why all the data arerepresented as percentages of the transactivating activity of thewild-type protein of the isolate ACH320.2A.2.1 and why the data obtainedfrom the transfection of the HL3T1 cells and the co-culture of the293T/HL3T1 cells are compared for each construction. As shown in FIG. 5,the R55L mutation does not significantly alter the transduction capacityof the protein. The mutation R52L significantly reduces the transductioncapacity of the protein (5-fold reduction in the CAT activity betweenthe transfected HL3T1 cells with this construction and the HL3T1 cellsco-cultivated with the 293T cells expressing pEGFP-TEX-R52L). The doublemutant R52L-R55L shows a complete loss of its transduction capacity, asshown by the background noise of the CAT activity measured afterco-culture. The results obtained both with the R52L and R52L-R55Lmutants suggest that Tat's transduction capacity is correlated with thenumber of arginine residues in the basic domain (27). It would seem thatresidue R55 is less important for the Tat's transduction capacity thanresidue R52. Therefore, the localization of the arginine residues couldalso play a role in the complete transduction mechanism.

EXAMPLE 5 Cloning of Cell Lines Transfected with the NucleotideSequences of the Invention

Numerous functional tests on the regulation of cell genes by the Tatprotein of HIV-1 involve the use of cell lines expressing this proteinin constitutive manner. The Inventors therefore established cell linesexpressing the different Tat-protein mutants. In order to do this, HeLacells were seeded at 2.5×10⁵ cells per well of a 6-well plate thentransfected the next day with 2 μg of DNA coding for the various Tatmutants using the reagent Exgen 500 (marketed by Euromedex). In order tocarry out a biological cloning of these transfected lines, the cellswere trypsinized and counted 3 days after transfection, then seeded at aconcentration of 3 to 30 cells per well in flat-bottomed 96-well plates,at a rate of 3 to 5 96-well plates per transfection (for a total of 288to 480 wells per transfection). The culture in 96-well plates was thencarried out for 15 days in the presence of 500 μg/ml of geneticin(Geneticin Sulfate, Gibco-BRL). After 15 days, the wells in which thecells were still alive and had multiplied notably were considered aspositive. In standard manner, a biological cloning was consideredsuccessful when each 96-well plate from the same transfection containedless than 10 positive wells per plate. From 3 to 15 positive wells pertransfection were then amplified in the presence of geneticin for 6passages in order to obtain a sufficient quantity of cells for freezing.On the sixth passage, the expression of the Tat protein was verified byimmunotransfer (western blot) for each clone.

After verification of the expression of Tat by immunotransfer in thecell lines thus generated, the Inventors used a plasmid constructioncontaining the CAT gene dependent on the viral HIV promoter (LTR-CATconstruction) in order to transfect the different clones of cell linesthus obtained. It was thus possible to show that the stable expressionof the Tat mutants in this cell line did not modify theirtransactivating activity (FIG. 6).

EXAMPLE 6 Mutant in Position 58

The single mutant S58A is obtained using as matrix the cDNA of thewild-type Tat gene of the strain ACH.320.2A.2.1 and as primer pair thepair S58A (M5′)/S58A (M3′). However, other HIV-1 strains exist whichnaturally carry an alanine in position 58 and which have all theproperties of a functional Tat (nuclear localization, transactivationetc.) such as for example the strain HXB2. Such a mutation S58A on thestrain ACH320.2A.2.1 does not modify the behaviour of the protein anddoes not make it possible to achieve the detoxification of Tat. TABLE 1Sequence End primers, 1st cycle E5′ 5′- GAA TTC ATG GAG CCA GTA GAT C-3′ E3′ 5′- AGA TCT CTA ATC GAC CGG ATC- 3′ End primers, 2nd cycle EcoR I5′- AAA GAA TTC ATG GAG CCA GTA GAT CC- 3′ E6854 5′- AAA GAT CTC TAA TCGACC GGA TCT GTC TCT GTC TC- 3′ Sal I 5′- AAG TCG ACC TAA TCG ACC GGA TCTGTC TCT GTC TC- 3′ Internal primers W11F (M5′) 5′- CCA GTA GAT CCT AAACTA GAG CCC TTC AAG CAT CCA G-3′ C27S (MS′) 5′- ACA ATT GCT ATT CGA AAAAGT G- 3′ C27S (M3′) 5′- CAC TTT TTC GAA TAG CAA TTG T- 3′ K50R (M5′)5′- ATC TCA TAT GGC AGG CGG AAG -3′ K50R (M3′) 5′- CTT CCG CCT GCC ATATGA GAT -3′ K51T (M5′) 5′- GGC AGG AAG ACC CGG AGA CAG C- 3′ K51T (M3′)5′- GCT GTC TCC GGG TCT TCC TGC C- 3′ R52L (M5′) 5′- GGC AGG AAG AAG CTTAGA CAG CGA CGA AGA TC -3′ R52L (M3′) 5′- GAT CTT CGT CGC TGT CTA AGCTTC TTC CTG CC- 3′ R55L (M5′) 5′- GGC AGG AAG AAG CGG AGA CAG CTG CGAAGA TC- 3′ R55L (M3′) 5′- GAT CTT CGC AGC TGT CTC CGC TTC TTC CTG CC- 3′R57L (M5′) 5′- GAC AGC GAC GAC TAT CTC CTC AAG AC -3′ R57L (M3′) 5′- GTCTTG AGG AGA TAG TCG TCG CTG TC- 3′ G79A (MS′) 5′- CAG CCC CGA GCG GATCCG ACA GG- 3′ G79A (M3′) 5′- CCT GTC GGA TCC GCT CGG GGC TG- 3′ K89L(M3′) 5′- CTG TCT CTG TCT CTC TCT CCA CCT TAA GCT TCG ATT CC- 3′ E92Q(M3′) 5′- CTG TCT CTG TCT CTC TTT GCA CCT TCT TCT TCG AAT CC- 3′R52L-R55L (M5′) 5′- GGC AGG AAG AAG CTT AGA CAG CTG CGA AGA TC - 3′R52L-R55L (M3′) 5′- GAT CTT CGC AGC TGT CTA AGC TTC TTC CTG CC - 3′R55L-R57L (M5′) 5′- GAA GCG GAG ACA GCT GCG ACT ATC TCC TCA AGA C -3′R55L-R57L (M3′) 5′- GTC TTG AGG AGA TAG TCG CAG CTG TCT CCG CTT C -3′S58A (M5′) 5′- GAC AGC GAC GAA GAG CAC CTC AAG ACA GT -3′ S58A (M3′) 5′-ACT GTC TTG AGG TGC TCT TCG TCG CTG TC -3′

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1. Detoxified and immunogenic mutant of the Tat protein of the HIV-1virus, characterized in that it comprises at least two mutations inregions 4 and/or 5 of the wild-type Tat protein, and in that, when themutation is in the region of domain 4, it is in the part delimited bythe amino acid in position 49 to the amino acid in position 57, and inthat, when the mutation is in domain 5, it is either in the RGD motif,or in the region 88-92, preferably in positions 89 and/or 92, themutations being mutations by substitution of one amino acid by another.2. Mutant according to claim 1, characterized in that it comprises atleast one mutation in region
 4. 3. Mutant according to claim 1,characterized in that the mutations in domains 4 and/or 5 are capable ofconferring at least one of the following properties: the cancellation ofthe transcellular effect of the wild-type Tat protein, the alteration ofthe nuclear localization of the wild-type Tat protein.
 4. Mutantaccording to claim 1, characterized in that it comprises an additionalmutation capable of conferring a loss of the transactivating activity ofthe wild-type Tat protein.
 5. Mutant according to claim 1, characterizedin that it comprises a mutation in the N-terminal region of domain 4 ofthe wild-type Tat protein in the part delimited by the amino acid inposition 49 to the amino acid in position
 55. 6. Mutant according toclaim 1, characterized in that it comprises a mutation in domain 2 ofthe wild-type Tat protein, in particular the replacement of any one ofthe cysteines, advantageously by a serine.
 7. Mutant according to claim1, characterized in that it comprises at least one of the followingmutations: replacement in position 27 of a cysteine by a serine,replacement in position 51 of a lysine by a threonine, replacement inposition 52 of an arginine by a leucine, replacement in position 55 ofan arginine by a leucine, replacement in position 57 of an arginine by aleucine, replacement in position 79 of a glycine by an alanine,replacement in position 89 of a lysine by a leucine, replacement inposition 92 of a glutamic acid by a glutamine.
 8. Mutant according toclaim 1, characterized in that it is chosen from the mutants having twomutations as indicated hereafter, each of the mutations beingrepresented by a triplet: letter-figure-letter, the figure of whichindicates the position of the mutated amino acid, the letter precedingthe figure corresponds to the amino acid to which the mutation relatesand the letter following the figure corresponds to the amino acidreplacing the amino acid preceding the figure: K51T-R52L (SEQ ID NO: 2)K51T-R55L (SEQ ID NO: 3) K51T-R57L (SEQ ID NO: 4) K51T-G79A (SEQ ID NO:5) K51T-K89L (SEQ ID NO: 6) K51T-E92Q (SEQ ID NO: 7) R52L-R55L (SEQ IDNO: 8) R52L-R57L (SEQ ID NO: 9) R52L-G79A (SEQ ID NO: 10) R52L-K89L (SEQID NO: 11) R52L-E92Q (SEQ ID NO: 12) R55L-R57L (SEQ ID NO: 13) R55L-G79A(SEQ ID NO: 14) R55L-K89L (SEQ ID NO: 15) R55L-E92Q (SEQ ID NO: 16)R57L-G79A (SEQ ID NO: 17) R57L-K89L (SEQ ID NO: 18) R57L-E92Q (SEQ IDNO: 19) G79A-K89L (SEQ ID NO: 20) G79A-E92Q (SEQ ID NO: 21) K89L-E92Q(SEQ ID NO: 22).


9. Mutant according to claim 8, characterized in that it is chosen fromthe following mutants: K51T-R55L (SEQ ID NO: 3) R52L-R55L (SEQ ID NO: 8)R52L-G79A (SEQ ID NO: 10) R55L-R57L (SEQ ID NO: 13) G79A-K89L (SEQ IDNO: 20).


10. Mutant according to claim 1, characterized in that it is chosen fromthe mutants having three mutations as indicated hereafter, each of themutations being represented by a triplet: letter-figure-letter, thefigure of which indicates the position of the mutated amino acid, theletter preceding the figure corresponds to the amino acid to which themutation relates and the letter following the figure corresponds to theamino acid replacing the amino acid preceding the figure: C27S-K51T-R52L(SEQ ID NO: 23) C27S-K51T-R55L (SEQ ID NO: 24) C27S-K51T-R57L (SEQ IDNO: 25) C27S-K51T-G79A (SEQ ID NO: 26) C27S-K51T-K89L (SEQ ID NO: 27)C27S-K51T-E92Q (SEQ ID NO: 28) C27S-R52L-R55L (SEQ ID NO: 29)C27S-R52L-R57L (SEQ ID NO: 30) C27S-R52L-G79A (SEQ ID NO: 31)C27S-R52L-K89L (SEQ ID NO: 32) C27S-R52L-E92Q (SEQ ID NO: 33)C27S-R55L-R57L (SEQ ID NO: 34) C27S-R55L-G79A (SEQ ID NO: 35)C27S-R55L-K89L (SEQ ID NO: 36) C27S-R55L-E92Q (SEQ ID NO: 37)C27S-R57L-G79A (SEQ ID NO: 38) C27S-R57L-K89L (SEQ ID NO: 39)C27S-R57L-E92Q (SEQ ID NO: 40) C27S-G79A-K89L (SEQ ID NO: 41)C27S-G79A-E92Q (SEQ ID NO: 42) C27S-K89L-E92Q (SEQ ID NO: 43).


11. Mutant according to claim 10, characterized in that it is chosenfrom the following mutants: C27S-K51T-R55L (SEQ ID NO: 24)C27S-R52L-R55L (SEQ ID NO: 29) C27S-R52L-G79A (SEQ ID NO: 31).


12. Mutant according to claim 1, characterized in that it is chosen fromthe mutants having four mutations as indicated hereafter, each of themutations being represented by a triplet: letter-figure-letter, thefigure of which indicates the position of the mutated amino acid, theletter preceding the figure corresponds to the amino acid to which themutation relates and the letter following the figure corresponds to theamino acid replacing the amino acid preceding the figure:C27S-K51T-R52L-G79A (SEQ ID NO: 44) C27S-K51T-R52L-K89L (SEQ ID NO: 45)C27S-K51T-R52L-E92Q (SEQ ID NO: 46) C27S-K51T-R55L-G79A (SEQ ID NO: 47)C27S-K51T-R55L-K89L (SEQ ID NO: 48) C27S-K51T-R55L-E92Q (SEQ ID NO: 49)C27S-K51T-R57L-G79A (SEQ ID NO: 50) C27S-K51T-R57L-K89L (SEQ ID NO: 51)C27S-K51T-R57L-E92Q (SEQ ID NO: 52) C27S-K51T-G79A-K89L (SEQ ID NO: 53)C27S-K51T-G79A-E92Q (SEQ ID NO: 54) C27S-K51T-K89L-E92Q (SEQ ID NO: 55)C27S-R52L-G79A-K89L (SEQ ID NO: 56) C27S-R52L-G79A-E92Q (SEQ ID NO: 57)C27S-R52L-K89L-E92Q (SEQ ID NO: 58) C27S-R52L-R55L-G79A (SEQ ID NO: 59)C27S-R52L-R55L-K89L (SEQ ID NO: 60) C27S-R52L-R55L-E92Q (SEQ ID NO: 61)C27S-R52L-R57L-G79A (SEQ ID NO: 62) C27S-R52L-R57L-K89L (SEQ ID NO: 63)C27S-R52L-R57L-E92Q (SEQ ID NO: 64) C27S-R55L-G79A-K89L (SEQ ID NO: 65)C27S-R55L-G79A-E92Q (SEQ ID NO: 66) C27S-R55L-K89L-E92Q (SEQ ID NO: 67)C27S-R55L-R57L-G79A (SEQ ID NO: 68) C27S-R55L-R57L-K89L (SEQ ID NO: 69)C27S-R55L-R57L-E92Q (SEQ ID NO: 70) C27S-R57L-G79A-K89L (SEQ ID NO: 71)C27S-R57L-G79A-E92Q (SEQ ID NO: 72) C27S-R57L-K89L-E92Q (SEQ ID NO: 73)C27S-G79A-K89L-E92Q (SEQ ID NO: 74).


13. Mutant according to claim 12, characterized in that it is chosenfrom the following mutants: C27S-K51T-R55L-G79A (SEQ ID NO: 47)C27S-K51T-R55L-K89L (SEQ ID NO: 48) C27S-K51T-R55L-E92Q (SEQ ID NO: 49)C27S-R52L-R55L-G79A (SEQ ID NO: 59).


14. Mutant according to claim 1, characterized in that it is chosen fromthe mutants having five mutations as indicated hereafter, each of themutations being represented by a triplet: letter-figure-letter, thefigure of which indicates the position of the mutated amino acid, theletter preceding the figure corresponds to the amino acid to which themutation relates and the letter following the figure corresponds to theamino acid replacing the amino acid preceding the figure:C27S-K51T-G79A-K89L-E92Q (SEQ ID NO: 75) C27S-K51T-R52L-R55L-G79A (SEQID NO: 76) C27S-K51T-R52L-R55L-K89L (SEQ ID NO: 77)C27S-K51T-R52L-R55L-E92Q (SEQ ID NO: 78) C27S-K51T-R52L-R57L-G79A (SEQID NO: 79) C27S-K51T-R52L-R57L-K89L (SEQ ID NO: 80)C27S-K51T-R52L-R57L-E92Q (SEQ ID NO: 81) C27S-K51T-R52L-G79A-K89L (SEQID NO: 82) C27S-K51T-R52L-G79A-E92Q (SEQ ID NO: 83)C27S-K51T-R52L-K89L-E92Q (SEQ ID NO: 84) C27S-K51T-R55L-R57L-G79A (SEQID NO: 85) C27S-K51T-R55L-R57L-K89L (SEQ ID NO: 86)C27S-K51T-R55L-R57L-E92Q (SEQ ID NO: 87) C27S-K51T-R55L-G79A-K89L (SEQID NO: 88) C27S-K51T-R55L-G79A-E92Q (SEQ ID NO: 89)C27S-K51T-R55L-K89L-E92Q (SEQ ID NO: 90) C27S-K51T-R57L-G79A-K89L (SEQID NO: 91) C27S-K51T-R57L-G79A-E92Q (SEQ ID NO: 92)C27S-K51T-R57L-K89L-E92Q (SEQ ID NO: 93) C27S-R52L-R55L-R57L-G79A (SEQID NO: 94) C27S-R52L-R55L-R57L-K89L (SEQ ID NO: 95)C27S-R52L-R55L-R57L-E92Q (SEQ ID NO: 96) C27S-R52L-R55L-G79A-K89L (SEQID NO: 97) C27S-R52L-R55L-G79A-E92Q (SEQ ID NO: 98)C27S-R52L-R55L-K89L-E92Q (SEQ ID NO: 99) C27S-R52L-R57L-G79A-K89L (SEQID NO: 100) C27S-R52L-R57L-G79A-E92Q (SEQ ID NO: 101)C27S-R52L-R57L-K89L-E92Q (SEQ ID NO: 102) C27S-R52L-G79A-K89L-E92Q (SEQID NO: 103) C27S-R55L-R57L-G79A-K89L (SEQ ID NO: 104)C27S-R55L-R57L-G79A-E92Q (SEQ ID NO: 105) C27S-R55L-R57L-K89L-E92Q (SEQID NO: 106) C27S-R55L-G79A-K89L-E92Q (SEQ ID NO: 107)C27S-R57L-G79A-K89L-E92Q (SEQ ID NO: 108).


15. Mutant according to claim 14, characterized in that it is chosenfrom the following mutants: C27S-K51T-R55L-G79A-K89L (SEQ ID NO: 88)C27S-K51T-R55L-G79A-E92Q (SEQ ID NO: 89).


16. Nucleotide sequences coding for one of the mutants according toclaim
 1. 17. Cell line transfected with a nucleotide sequence accordingto claim
 16. 18. Antibodies directed against one of the mutantsaccording to claim 1, not recognizing domain D1 of the wild-typeprotein.
 19. Antibodies according to claim 18, recognizing the wild-typeprotein.
 20. Antibodies according to claim 18, not recognizing thewild-type protein.
 21. Pharmaceutical composition, in particular avaccine, containing as active ingredient at least one of the mutantsaccording to claim 1, placed under the control of elements necessary toa constitutive expression of one of the mutants, in combination with apharmaceutically suitable vehicle.
 22. Diagnostic composition for thedetection and/or quantification of HIV-1 virus comprising at least onemutant as defined in claim
 1. 23. Process for the detection and/orquantification of the HIV-1 virus in a biological sample taken from anindividual capable of being infected with HIV-1, such as plasma, serumor tissue, characterized in that it comprises stages consisting of:bringing said biological sample into contact with a diagnosticcomposition comprising a mutant as defined in claim 1, underpredetermined conditions which allow, if necessary, the formation ofantibody/antigen complexes between the mutant defined above andantibodies directed against the wild-type Tat protein or between theantibodies defined above and the wild-type Tat protein, and detectingand/or quantifying the formation of said complexes by any appropriatemeans.
 24. Method for the in vitro diagnosis of the HIV-1 virus in abiological specimen or sample, which comprises using at least one mutantas defined in claim
 1. 25. Method for the preparation of a vaccinecomposition, which comprises using at least one mutant as defined inclaim 1.